Spinal muscular atrophy (SMA) is an inherited neurodegenerative disease characterized by motor neuron loss and skeletal muscle atrophy. SMA is the most common genetic cause of death in infancy, but no effective treatment is currently available. Although it is well established that reduced levels of the survival motor neuron (SMN) protein due to homozygous mutations in the SMN1 gene cause SMA, the molecular basis of motor neuron dysfunction is unknown. The identification of molecules that are affected by SMN deficiency and contribute to SMA pathology is critically needed not only for elucidation of disease mechanisms but also for development of effective therapies. In previous studies, we have identified a novel, evolutionarily conserved transmembrane protein-which we named Stasimon-whose expression is decreased by SMN deficiency and that contributes to SMN-dependent motor neuron dysfunction in Drosophila and zebrafish models of SMA. Since these animal models do not have the low steady levels of SMN characteristic of SMA patients, this project will investigate whether decreased Stasimon function contributes to SMA pathology in a mouse model that more closely resemble the human disease. To date, SMN target genes with a demonstrated role in the pathogenesis of SMA mice have not been described. In Aim 1, we will analyze the effects of SMN deficiency on Stasimon expression at the mRNA and protein levels in tissues of SMA mice, with a particular focus on the spinal cord. Laser capture microdissection will be employed to isolate selected, disease-relevant neuronal types from control and SMA mice for RNA analysis. These will include motor neurons (MNs) in the ventral horns of the lumbar spinal cord and proprioceptive neurons located in the dorsal root ganglia (DRG). Changes in Stasimon expression will be investigated in MNs from different motor columns (lateral and medial) as well as distinct lumbar segments that are differentially affected by SMN deficiency in a time-dependent manner. Immunohistochemistry will be used to define the in vivo distribution of Stasimon protein under normal conditions and any changes caused by SMN deficiency in the same neural types. In Aim 2, we will study the effect of increasing Stasimon expression on the phenotype of SMA mice in order to assess its involvement in SMA pathology. Recent studies demonstrated that increasing SMN levels postnatally through systemic injection of an adeno-associated virus (AAV9) expressing SMN from a ubiquitous promoter rescues the SMA phenotype in a severe mouse model. We will use this previously validated, AAV9-mediated gene delivery system for expression of Stasimon in SMA mice. A comprehensive set of assays will then be carried out to monitor Stasimon-dependent improvement in morphological and functional parameters of sensory-motor circuit connectivity that are severely compromised in SMA. Collectively, these experiments have the potential to identify Stasimon as a downstream target of SMN dysfunction that contributes to SMA pathology in a mouse model of this devastating human disease. PUBLIC HEALTH RELEVANCE: SMA is an incurable motor neuron disease and the leading genetic cause of death in infancy. We will establish whether a novel gene we have identified for its potential involvement in the disease process has a direct role in the neuromuscular pathology of a mouse model of SMA. If successful, these studies will reveal an important molecule for elucidation of the molecular mechanisms of SMA as well as a candidate target for development of new therapeutic strategies for this genetic disorder.